Project Summary/Abstract A normal and robust immune system relies on the development of a diverse repertoire of T cells that are tolerant of self-tissues. The thymus is a critical site for the development and education of T cell to promote tolerance to self and thus prevent autoimmune diseases, such as Type 1 diabetes or multiple sclerosis. Within the thymus, the Autoimmune Regulator (Aire) gene is a key player in the maintenance of immune tolerance as evident by its identification as the defective gene in the human autoimmune syndrome Autoimmune Polyglandular Syndrome Type 1. Aire acts within specialized medullary thymic epithelial cells (mTECs) to promote the expression of hundreds of self-antigens for the purpose of removing developing self-reactive T cells in a process known as negative selection. Recently however, we have uncovered new populations of mTECs that develop from cells that formerly expressed Aire that we have termed post-Aire-expressing (post- Aire) cells. These cells appear to fall into 2 subsets: (1) a keratinocyte-like cell that is associated with Hassall's corpuscles and (2) a tuft-like cell similar to specialized intestinal tuft cells. These cells show distinct patterns of gene expression with intermediate levels of self-antigen expression compared to conventional mTECs, suggesting that they may play a distinct and complementary role in mediating T cell tolerance in the thymus. We have developed a powerful set of genetic tools and mouse reporter lines that allows us to mark, follow and purify these unique cells in order to study their function in the thymus and their contribution to immune tolerance. We hypothesize that post-Aire cells in the thymus represent a unique subset of thymic epithelial cells that mediate the maturation and development of tolerogenic T cell populations. Therefore, we propose to test this hypothesis through the following specific aims: (1) Define markers of cell identity and key pathways of cell development in thymic tuft cells, (2) Examine the effect of post- Aire mTEC's on T cell selection and thymocyte development, and (3) Assess the contributions of post- Aire mTECs to immune tolerance in vivo. These studies will allow us to uncover the development and function of these novel cells within the thymus as well as their contribution to T cell selection and maturation as well as induction of other important immune regulators, such as T regulatory and invariant natural killer cells. In this way, we hope to understand the role of these cells in the promoting immune tolerance and how they may be employed in the prevention of autoimmune disease.